Mycoplasma pneumoniae CARDS toxin mediated ADP-ribosylation of NLRP3 inflammasome

 描述（由申请人提供）：拟议的项目提出了关于肺炎支原体（Mp）及其新发现的ADP-核糖基化、空泡化毒素（指定为社区获得性呼吸窘迫综合征（ARDS）毒素）在气道疾病发病机制中所起作用的基本和应用问题。CARDS毒素显着概括了伴随Mp感染的促炎细胞因子/趋化因子谱和组织病理学。此外，Mp毒素的失活显著减少了Mp感染期间成熟形式白细胞介素-1 β（IL-1 β）的释放。IL-1 β是一种重要的促炎细胞因子，其决定了与广泛的炎性疾病相关的炎症的严重程度。活性成熟IL-1 β的释放是通过炎性小体复合物介导的半胱天冬酶-1激活完成的，半胱天冬酶-1是负责将未成熟的pro-IL-1 β裂解成其成熟形式的酶。NLRP 3是炎性体复合物的关键组分，并且多种信号和刺激触发NLRP 3炎性体复合物的形成。在气道中，Mp和Mp毒素主要在肺泡巨噬细胞中检测到，我们的初步研究表明，在初级巨噬细胞中，Mp毒素激活NLRP 3炎性小体，导致IL-1 β产生。如通过免疫共沉淀分析推断的，在原代巨噬细胞中，肉毒毒素与NLRP 3炎性体共定位，并与NLRP 3相互作用。令人惊讶的是，我们的初步研究证明了NLRP 3的腺苷二磷酸核糖基化是由肉毒毒素介导的，并且NLRP 3的活化需要肉毒毒素的腺苷二磷酸核糖基转移酶（ART）活性。基于我们的初步结果，我们假设Mp和特别是Mp毒素是气道功能障碍和炎症的关键介质，通过Mp毒素ART相关机制，涉及NLRP 3炎性小体激活和气道中活性成熟IL-1 β的释放。我们计划通过以下步骤来检验这一假设：a）研究NLRP 3炎性体组装和IL-1 β分泌中NLRP 3 ADP核糖基化和结合活性的作用，B）检查NLRP 3在NLRP 3介导的IL-1 β产生中的体内参与，c）研究IL-1 β在触发NLRP 3介导的肺部炎性疾病病理中的作用，和d）阐明在体内IL-1 β产生和炎性小体活化过程中，α毒素的ART活性的作用。我们的长期目标是制定有效的策略，以诊断，治疗和预防大量儿童和成人人群中的MP相关气道疾病。了解炎性小体通过Mp毒素激活的机制应导致治疗干预和改善许多患有与Mp感染相关的急性和慢性气道和肺外病理的个体的健康状况。所提出的研究还将提供关于炎性小体激活机制的新见解，因为炎性小体的翻译后ADP-核糖基转移酶介导的修饰是炎性小体激活的新机制，随后释放IL-1 β和相关病理。

 DESCRIPTION (provided by applicant): The proposed project asks fundamental and applied questions concerning the role that Mycoplasma pneumoniae (Mp) and its newly discovered ADP-ribosylating, vacuolating toxin, designated Community Acquired Respiratory Distress Syndrome (CARDS) toxin, play in the pathogenesis of airway disorders. CARDS toxin remarkably recapitulates the pro-inflammatory cytokine/chemokine profiles and histopathology that accompany Mp infection. In addition, inactivation of CARDS toxin significantly reduced the release of mature form interleukin-1ß (IL-1ß) during Mp infection. IL-1ß is a critical pro-inflammatory cytokine that dictates severity of inflammation associated with a wide spectrum of inflammatory diseases. Release of active mature IL-1ß is accomplished by inflammasome complex-mediated activation of caspase-1, the enzyme responsible for cleaving immature pro-IL-1ß into its mature form. NLRP3 is a key component of the inflammasome complex, and multiple signals and stimuli trigger formation of the NLRP3 inflammasome complex. In the airway, Mp and CARDS toxin are primarily detected in the alveolar macrophages, and our preliminary studies show NLRP3 inflammasome activation by CARDS toxin in primary macrophages leading to IL-1ß production. CARDS toxin co-localized with NLRP3 inflammasome in primary macrophages and interacted with NLRP3 as deduced by co-immunopreciptation analysis. Surprisingly, our preliminary studies demonstrated CARDS toxin-mediated ADP-ribosylation of NLRP3 and a requirement of ADP- ribosyltransferase (ART) activity of CARDS toxin for NLRP3 activation. Based on our preliminary results, we hypothesize that Mp and specifically CARDS toxin are key mediators of airway dysfunction and inflammation via CARDS toxin ART-related mechanisms involving NLRP3 inflammasome activation and resulting release of active mature IL-1ß in the airway. We plan to test this hypothesis by - a) studying the role of CARDS toxin ADP ribosylation and binding activities in NLRP3 inflammasome assembly and IL-1ß secretion, b) examining in vivo involvement of NLRP3 in CARDS toxin mediated IL-1ß production, c) investigating the role of IL- in triggering CARDS toxin-mediated lung inflammatory disease pathology, and d) elucidating the role of ART activity of CARDS toxin during in vivo IL-1ß production and inflammasome activation. Our long-term goal is to develop effective strategies to diagnose, treat and prevent Mp-related airway diseases in a substantial population of both children and adults. Understanding the mechanisms by which the inflammasome is activated via CARDS toxin should lead to therapeutic interventions and improved health in many individuals who suffer from acute and chronic airway and extrapulmonary pathologies linked to Mp infections. The proposed studies will also provide new insights about inflammasome activation mechanisms since post- translational ADP-ribosyltransferase-mediated modification of the inflammasome is a novel mechanism for inflammasome activation with subsequent release of IL-1ß and associated pathologies.